Hello All,
Both abcam and biorad protocols state that low methanol concentration in transfer buffer could swell gel pore and this can increase transfer efficiency of protein. So I just wonder that if using transfer buffer without both methanol and sds, Can it swell the gel pore (maybe in during 5-15 min equilibration or wet transfer) and lose out/leak some small protein on gel (eg.
Although many companies now suggest buffers without methanol, 20% (but at least 10%) is quite critical in Western transfer buffers using traditional methods. I know from personal experience. Also, SDS in the transfer buffer can make a huge difference. Add SDS at 0.37g /LITRE.
You have to equilibrate the gel with methanol buffer since that is the whole point of equilibration - to allow the gel to completely exchange gel buffer with the transfer buffer. Also, the blotting papers and membrane.
Good luck !
I agree with Prem's response and would like to add that the presence of methanol appears to allow efficient transfer and retention of small molecular weight (70 kDa) on the PVDF membrane.
Hi Asa, PVDF membranes bind proteins mostly by hydrophobic interactions and not by pore size.
Smaller proteins in gels are generally transferred faster than larger proteins because they are less restrained by the polyacrylamide gel matrix. It is more pronounced if you are using a uniform % gel than a gradient gel.
Anyway, try reducing the time of transfer.
Hi Asa,
I agree with all the above comments. I used 20% MeOH for transfer of a 6 kDa protein and I reduce the transfer time to half 'in a uniform % gel) of what I normally use for a 40 kDa protein and it works fine. Recently I'm working with gradient gels (4-20%) and there I also use 20% MeOH and the same transfer time as for larger proteins and I got similar results as before.
Good luck!
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